The Effects of Antipsychotics in Experimental Models of Krabbe Disease

The role of altered myelin in the onset and development of schizophrenia and changes in myelin due to antipsychotics remains unclear. Antipsychotics are D2 receptor antagonists, yet D2 receptor agonists increase oligodendrocyte progenitor numbers and limit oligodendrocyte injury. Conflicting studies suggest these drugs promote the differentiation of neural progenitors to oligodendrocyte lineage, while others report antipsychotics inhibit the proliferation and differentiation of oligodendrocyte precursors. Here, we utilised in-vitro (human astrocytes), ex-vivo (organotypic slice cultures) and in-vivo (twitcher mouse model) experimental study designs of psychosine-induced demyelination, a toxin that accumulates in Krabbe disease (KD), to investigate direct effects of antipsychotics on glial cell dysfunction and demyelination. Typical and atypical antipsychotics, and selective D2 and 5HT2A receptor antagonists, attenuated psychosine-induced cell viability, toxicity, and morphological aberrations in human astrocyte cultures. Haloperidol and clozapine reduced psychosine-induced demyelination in mouse organotypic cerebellar slices. These drugs also attenuated the effects of psychosine on astrocytes and microglia and restored non-phosphorylated neurofilament levels, indicating neuroprotective effects. In the demyelinating twitcher mouse model of KD, haloperidol improved mobility and significantly increased the survival of these animals. Overall, this study suggests that antipsychotics directly regulate glial cell dysfunction and exert a protective effect on myelin loss. This work also points toward the potential use of these pharmacological agents in KD.


Introduction
Schizophrenia is a chronic condition involving the complex interplay between genetic and environmental factors and is thought to be associated with aberrant neurodevelopment [1,2]. Schizophrenia is associated with abnormalities in grey and white matter, as well as loss of cerebral volume and anatomical pathology [3]. While neuronal dysfunction has been well studied in schizophrenia, the role of glial cells in the pathophysiology of this disease is still emerging, and, in particular, the role of altered oligodendrocyte biology is less clear [4]. Differing theories exist, although there is no consensus as to whether altered levels of myelin are linked with the onset of this illness, if altered myelination is a cause or consequence associated with the development of the condition, and/or how levels of myelin change during short and long-term use of antipsychotic treatment. There is an agreement, however, that patients with schizophrenia have decreased white matter volume and integrity, where post-mortem studies demonstrate white matter pathology linked to deficits in myelin, axons and mature oligodendrocytes [5,6]. In addition, altered expression and risk variants of several genes playing a role in oligodendrocyte maturation and myelination have also been associated with this illness, where variants in myelin-related genes increase schizophrenia susceptibility [7][8][9]. in DMSO. Working concentrations from stock compounds were made using serum-free media prior to treatments. Primary antibodies (1:1000 dilution) were mouse anti-vimentin (Vimentin, Santa-Cruz, Heidelberg, Germany; Sc-373717; AB10917747), chicken anti-glial fibrillary acidic protein (GFAP, Abcam, Cambridge, UK; ab4674; AB304558), mouse antimyelin oligodendrocyte glycoprotein (MOG, Millipore, Darmstadt, Germany; MAB5680; AB1587278), rabbit anti-myelin basic protein (MBP, Abcam, Cambridge, UK; ab40390; AB1141521), chicken anti-neurofilament H (NFH, Millipore, Darmstadt, Germany; ab5539; AB177520), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1, Wako, Neuss, Germany; 019-19741; AB839504) and mouse anti-neurofilament H non-phosphorylated (anti-SMI32, Millipore, Darmstadt, Germany; NE1023; AB2715852). Secondary antibodies (1:1000 dilution) were goat Alexa-549 anti-mouse (Jackson immune research, Cambridge, UK; 115-5060-068), goat Alexa-633 anti-chicken (Invitrogen, Waltham, MA, USA; A21103) and goat Alexa-488 anti-rabbit (Invitrogen, Waltham, MA, USA; A11008). Hoescht (4 µM) was used to stain cell nuclei (Thermo Scientific, Waltham, MA, USA; 62249).

Human Astrocytes Cell Culture Studies
Human astrocytes (ScienCell Research Laboratory, Carlsbad, CA, USA; Cat No. #1800, Lot No. 9063) were cultured exactly as we have described previously [19][20][21][22][23][24][25][26][27][28]. Confluent cells were cultured in serum-free media for four hours and then treated as specified in Figure legends. For MTT and LDH assays, human astrocytes were seeded in 96 well plates at a density of 0.01 × 10 6 per well and cultured for 24 h until greater than 80% confluent. For MTT assays, after treatment, media was removed and replaced with 100 µL of fresh serum-free media supplemented with 10 µL of 12 mM MTT Formazan (Sigma, Darmstadt, Germany; m2003) and plates were incubated for 2.5 h at 37 • C. Subsequently, 75 µL of media was removed, and 50 µL of DMSO was added per well. Cells were incubated for ten minutes, and the absorbance was read at 540 nm. For LDH assays, cytotoxicity was measured on aliquots of the cellular supernatant using the CyQUANTTM LDH Cytotoxicity Assay Kit following manufacturer instructions. LDH activity was measured using 490-nm absorbance. The immunocytochemistry and fluorescent microscopy of human astrocytes was carried out as we have outlined previously [19][20][21][22][23][24][25][26][27][28]. Images of cells were acquired using scanning confocal microscopy (Leica, Ashbourne, Ireland; SP8) at 10× and 20× magnification. The number of astrocyte projections of 20-30 cells per treatment group was analysed using ImageJ software.

Mouse Organotypic Cerebellar Slice Culture Studies
Organotypic cerebellar slice cultures were prepared exactly as we have described previously [21,[29][30][31]. At 12 days in vitro (DIV), slices were treated as per Figure legends, and slices were prepared for immunohistochemistry at 14 DIV. Immunohistochemistry and confocal fluorescent microscopy were also carried out, as we have outlined previously [19][20][21][22]24,26,28,30,32]. Cerebellar slices were washed with PBS and then fixed with 4% PFA for 10 min. Slices were then blocked and permeabilised with 10% BSA and 0.05% triton-x in PBS overnight. Slices were then incubated for 48 h in an appropriate primary antibody diluted in 2% BSA + 0.1% triton-x and then washed and incubated for 24 h in an appropriate secondary antibody. The slices were rinsed and placed on glass microscope slides with SlowFade ® (Invitrogen, Waltham, MA, USA; s36936), and the edges were closed with varnish. Slides were stored in the dark until imaged. Images of slices were acquired using scanning confocal microscopy (Leica, Ashbourne, Ireland; SP8) at 10× and 20× magnification. The mean fluorescence for each treatment group was analysed by examining regions of interest (ROI). Image analysis was carried out using ImageJ (https://imagej.nih.gov/ij/) and Imaris ® software.

Twitcher Model In Vivo Studies
All animal work was carried out in compliance with EU legislation approved by Trinity College Dublin ethics committee and in accordance with guidelines from the Health Products Regulatory Authority (HPRA) under project authorisation number AE19136/P123. A colony of heterozygous twitcher mice obtained from Jackson Laboratory, Cambridge, UK (B6.CE-Galctwi/J Stock no:000845) were maintained for breeding under pathogen-free conditions. Ear punch samples were genotyped by TransnetYX (www.transnetyx.com) using real-time PCR. Haloperidol was given at a dose of 1 mg/kg/day in drinking water. Mice were kept in grouped cages, had constant access to food and water and were under a 12-h light/dark timetable. Humane endpoints, weight measurements, behavioural observations, twitching, immobility and locomotor scores were all as per established protocols previously published by our group [18]. Blinding was not conducted/possible for drug treatment or genotype as twitcher mice phenotype became observable over time. Data were pseudo-anonymised by using animal numbers, and analysis was carried out blinded to genotype and treatment. For open field maze (OFM) testing, animals were habituated on post-natal day (PND) 21 and 22 for 5 min. On PND 25, 28 and 30, animals were tested before the emergence of severe twitching or immobility. Animals were placed in the centre of the OFM apparatus, a 44 cm × 44 cm cage with highly darkened walls, for 5 min. Video recordings were analysed using ANYmaze tracking software (Stoelting), examining; distance (m), mean speed (m/s), max speed (m/s), time mobile (%), centre entries and corner time (%).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA), and the values shown are means +/− standard error of the mean. The sample size was calculated for one-way ANOVA by referencing previously published astrocyte psychosine toxicity experiments by our group [21,23,24,31]. A range of estimated mean change and standard deviation was set as 20-25% and 5-8%, respectively. A power of 0.8 and a stricter than conventional alpha of 0.1 was utilised to ensure experiments were not underpowered. Each experimental replicate or "n" for cultured human astrocytes were cells at different passages performed in triplicate. Normality was assessed by generating normal QQ plots for data and assessing for any obvious skewness. Clear outliers where identified were removed. Formal normality tests were carried out using D'Agostino-Pearson or Anderson-Darling tests for experiments with sufficient 'n' numbers. In experiments with smaller but still suitably powered sample sizes, normality was assessed using Shapiro-Wilk or Kolmogorov-Smirnov tests. Parametric one-way or two-way ANOVA tests or non-parametric Kruskal-Wallis tests followed by Tukey's or Dunn's multiple comparisons tests, respectively, were carried out for experiments comparing means from selected groups. Survival analysis was carried out using the Kaplan-Meier curve with a log-rank Mantel-Cox test. Further information on statistical methods is given in the results section and in Figure  legends. In all cases, the significance levels (alpha) were fixed at p < 0.05 *, p < 0.01 **, p < 0.001 *** and p < 0.0001 ****.

Haloperidol and Clozapine Attenuate Psychosine Induced Demyelination in Slice Cultures
Organotypic cerebellar slices were prepared from 10-day-old (P10) C57BL/6J mice, cultured for 12 DIV and treated with or without psychosine in the presence or absence of haloperidol or clozapine, with analysis conducted at 14 DIV (Figure 3a). Psychosine caused a concentration-dependent decrease, in both myelin markers MOG (Figure 3b,c) and MBP (Figure 3b,d), with a loss of NFH only at a high 1 µM concentration (Figure 3b,e). The psychosine (100nM) induced decrease in mean MOG and MBP fluorescence was attenuated by haloperidol 10 µM (Figure 3f-i) and Clozapine 10 µM (Figure 3j-m). In agreement with cellular studies (Figure 1), psychosine also showed a concentration-dependent decrease in the mean fluorescence of GFAP (Figure 4a (Figure 4l,m), with no effect of haloperidol (Figure 4j,l) or clozapine (Figure 4k,m). Psychosine caused axonal damage in organotypic cerebellar slices as demonstrated by a concentration-dependent increase in mean SMI-32 fluorescence, specifically within white matter tracts (Figure 5a,b), with no global change in levels across the whole cerebellar slice (Figure 5c). The psychosine 100nM induced increase in mean SMI-32 fluorescence was attenuated by haloperidol 10 µM (Figure 5a,d) and clozapine 10 µM (Figure 5a,f), again specifically in the white matter tracts with no change across the whole cerebellar slice (Figure 5e,g).

Haloperidol Improves Survival in Twitcher Mice
The typical antipsychotic haloperidol was given at a dose of 1 mg/kg/day to twitcher mice starting from PND5 onwards (Figure 6a). Haloperidol-treated twitcher mice had significantly increased body weight compared to untreated twitcher mice (Figure 6b). Twitching severity scores improved with the administration of haloperidol, showing a significantly slower progression of this behaviour over the course of the experiment (Figure 6c). Additionally, mobility scores deteriorated significantly less rapidly in haloperidol-treated twitcher mice compared to untreated twitcher mice during the experimental period ( Figure 6d). As expected, no significant differences in twitching scores, mobility scores and body weight were observed in wild-type control of haloperidol treated animals. An open field maze test was conducted on PND 25,28,30 when twitching and immobility scores were expected to be mild to moderate. A computer video tracking system (ANYmaze-Stoelting) was utilised to record traces (Figure 6e     psychosine-induced decrease in (d,e) GFAP and (d,f) Vimentin fluorescence at 100 nM psychosine. Clozapine 10 µM attenuated the psychosine-induced decrease in (g,h) GFAP and (g,i) Vimentin fluorescence at 100 nM psychosine. Psychosine in the presence or absence of (j,l) Haloperidol or (k,m) Clozapine did not alter (j,k) Iba1 fluorescence or (l,m) microglia morphology in white matter tracts or whole cerebellar slices areas. Confocal images at 10× and 20× magnification, scale bar 100 µm and 10 µm, respectively. Data are shown as mean +/− SEM, Kruskal-Wallis test, Dunn's multiple comparisons tests, #### p < 0.0001, **** p < 0.0001 and ** p < 0.01 (n = 5).

Haloperidol Improves Survival in Twitcher Mice
The typical antipsychotic haloperidol was given at a dose of 1 mg/kg/day to twitcher mice starting from PND5 onwards (Figure 6a). Haloperidol-treated twitcher mice had significantly increased body weight compared to untreated twitcher mice (Figure 6b).  Behavioural Metrics at PND25-No significant difference was observed between haloperidol-treated twitcher mice and untreated twitcher mice in the distance, mean speed, max speed, time mobile, centre entries or corner time. Additionally, no significant difference was observed in haloperidol-treated and untreated wild-type mice. Significant differences were observed between twitcher mice and wild-type littermates. Behavioural Metrics at PND28-Significant differences were observed between haloperidol-treated twitcher mice and untreated twitcher mice in distance, mean speed, time mobile, centre entries or corner time. No significant difference was observed for max speed between haloperidol-treated and untreated twitcher mice. No significant difference was observed in haloperidol-treated and untreated wild-type mice. Significant differences were observed between twitcher mice and wild-type littermates. Behavioural Metrics at PND30-Significant differences were observed between haloperidol-treated twitcher mice and untreated twitcher mice in distance, mean speed, time mobile, centre entries or corner time. No significant difference was observed for max speed between haloperidol-treated and untreated twitcher mice. No significant difference was observed in haloperidol-treated and untreated wild-type mice. Significant differences were observed between twitcher mice and wild-type littermates. One-way ANOVA followed by Tukey's multiple comparisons analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 8 per group).

The Regulation of Glia Cells by Antipsychotics
Structural and functional abnormalities in astrocytes, oligodendrocytes, and microglia, as well as glial progenitor cells, have been proposed in Schizophrenia [4,33]. Antipsychotics reduce grey and white matter and regulate all glial cell types, although some contradictory studies are noted, including: (i) Oligodendrocytes express D 2 receptors [13], with dopamine regulating myelin formation and the development and function of oligodendrocytes [14,15]. While agonists of D 2 receptors increase oligodendrocyte progenitor cell numbers and protect oligodendrocytes against oxidative injury [13], the D 2 antagonists haloperidol and clozapine are also shown to prevent apoptotic cell death in oligodendrocytes cultured under glucose deprived conditions [17]. Haloperidol and clozapine are protective in the inflammatory demyelinating EAE model of multiple sclerosis and the non-immune cuprizone model of demyelination [10,11]. (ii) Astrocyte subtypes are also regulated by antipsychotics [34], which enhance astroglial glutamatergic transmission [35], regulate cytokine expression [36], activate Cx43 channel activity [37] and alter a number of other signalling pathways in these cells. Antipsychotics improve disturbed metabolism in schizophrenia via dopamine receptors [38], enhance the release of D-serine from astrocytes [39] and reduce glutamate uptake in these cells [40]. Contrastingly, some studies highlight the concern that chronic antipsychotic use may contribute to progressive grey matter loss, perhaps by preferentially targeting astrocytes [41]. (iii) Microglia activation and release of inflammatory mediators is regulated by typical and atypical drugs [42][43][44]. Animal studies are controversial, with some suggesting antipsychotics increase Iba1 expression [45], while others show reduced microglial activation [46]. Lastly, drugs aiming to reduce microglial activation (minocycline) [47] and cyclooxygenase-2 (COX-2) inhibitors [48] have been proposed as effective treatment strategies for schizophrenia.

Psychosine Toxicity as a Model of Glial Cell Dysfunction
In this study, we used psychosine-induced in vitro and in vivo models to investigate the effects of antipsychotics on glial cell dysfunction and demyelination. Psychosine is a toxin that aggregates in the brains of those afflicted with the neurodegenerative disorder globoid cell leukodystrophy, Krabbe disease (KD) [49,50]. KD is a rare condition associated with progressive demyelination that mainly presents during infancy, but juvenile and adult presentations are possible. The mechanisms by which psychosine induces demyelination remain unclear; however, they may include (i) apoptotic processes and caspasedependent pathway activation [51][52][53][54][55], (ii) accumulation in lipid rafts associated with regional cholesterol increases and inhibition of PKC activity [49,[56][57][58] (iii) generation of LPC and arachidonic acid with the regulation of secreted phospholipase A2 (sPLA2) [52], and/or (iv) phosphorylation of neurofilament proteins reducing radial growth of axons, axonal defects and neuronal cell death [59,60]. Psychosine also negatively affects astrocyte viability, possibly via apoptotic processes and is also proposed to be pro-inflammatory [33,35,36,52]. It is possible that astrocytic reactivity may contribute a central role to the pathogenesis of KD [61], supported by data showing that oligodendrocytes from twitcher mice can myelinate axons when transplanted into the shiverer mouse model of demyelination [62].

Limitations and Future Directions
We acknowledge that our in-vitro human astrocyte studies relied on MTT assays of cell viability, and there are confounding factors that can influence such assays [63]. To address this and to improve the robustness of the data, we carried out additional LDH assays of cell toxicity, as well as morphological studies. We note, however, that future studies could examine the potential mechanisms by which antipsychotics might exert the protective effects seen, e.g., the use of pertussis toxin, selective protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) inhibitors could be utilised to further analyse the potential downstream signalling effects of antagonism of these receptors on psychosine induced toxicity in human astrocytes. Our ex-vivo slice culture experiments used the cerebellum, as it is rich in white matter and myelin-producing oligodendrocytes, and this area has been shown to be prone to psychosine-induced toxicity in previous studies [21,23,24,29]. In future studies, however, the use of other brain regions, as well as investigation of non-neuronal cells, such as inflammatory adaptive and innate immune cells, as well as cell-cell interactions, would be interesting. We also note that our twitcher mice invivo experiment used haloperidol, and future studies could use other antipsychotics to add weight to the suggestion here that antipsychotics may promote survival in twitcher mice and be a potential novel therapy for KD. Further studies, and in particular, neuroimaging studies using antipsychotics or drugs with similar pharmacology in animal models of KD, may highlight their possible use in future human trials. Lastly, we note that the twitcher mouse model used in this study is severe, modelling infantile KD, where antipsychotics may initially limit or slow demyelination, but where fatality ensues ultimately likely due to multi-organ toxicity caused by high concentrations of psychosine. The investigation of novel therapies in late-onset disease may be of interest, as well as combinatorial approaches that include limiting the production of psychosine, enhancement of its removal, as well as protecting against demyelination.

Conclusions
Here we show that psychosine (galactosylsphingosine) induces cell toxicity and reduces cell viability in human astrocytes, in agreement with previous studies [21,23,24,29]. We demonstrate that typical and atypical antipsychotics attenuate this psychosine-induced cell toxicity, cell viability and morphological changes in human astrocytes. Pharmacological analysis shows antipsychotics commonly act as antagonists or inverse agonists of dopamine and/or serotonin receptors. Our data show that both the selective D 2 antagonist (Eticlopride) and 5HT 2A antagonist (Volinanserin) reduce psychosine-induced toxicity and morphological changes in human astrocytes, with inhibition at D 2 receptors showing stronger efficacy. We also show that haloperidol and clozapine attenuate psychosine-induced demyelination in cultured organotypic cerebellar slices and reduce psychosine-induced loss of GFAP and vimentin expression. Regarding microglia, in agreement with previous studies [21,23,24,29], we saw little effect of psychosine on Iba1 expression, although we note Iba1 is not a specific marker of altered microglia reactivity. Investigating neuronal and axonal damage, we observed psychosine increases the expression of SMI-32 in the white matter axonal tracts of the arbour vitae, in agreement with our previous studies [21,23,24,29]. Haloperidol and clozapine treatment prevent the axonal expression of SMI-32, thus demonstrating a level of protection against psychosine-induced axonal damage in white matter tracts of cultured cerebellar slices. Lastly, to translate these findings into an in vivo setting, we show that haloperidol ameliorates weight loss, improves twitching, immobility and locomotor metrics in twitcher mice, and that haloperidol improves the survival of twitcher mice. To our knowledge, this study is the first to examine the direct effects of such a broad selection of commonly prescribed typical and atypical antipsychotics in both in vitro and in vivo models of psychosine-induced toxicity and demyelination.
The main points presented here are the following: (i) antipsychotics with dopamine and serotonin antagonism directly regulate glial cell dysfunction and exert a protective effect on myelin loss in cell cultures and organotypic cerebellar slices, and (ii) haloperidol, an antipsychotic with mainly dopamine antagonism improved survival and had a positive effect on behaviour in twitcher mice, an established demyelinating murine model of KD. Taken altogether, our findings indicate that antipsychotics are myelin protective and suggest the potential therapeutic benefit of agents with similar antipsychotic pharmacology in Krabbe disease. Institutional Review Board Statement: All experiments were conducted in accordance with EU guidelines and authorised by the Health Products Regulatory Authority, Ireland (HPRA).

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.